Haemophilus influenzae is a naturally transformable gram-negative bacterial pathogen that colonizes both the upper and lower human respiratory tracts. In the upper respiratory tract it can colonize the nasopharynx and then spread locally to cause disease in the ears (otitis media), sinuses (sinusitis), and meninges (meningitis). In the lower respiratory tract H. influenzae is associated with chronic obstructive pulmonary disease (COPD), bronchitis, and pneumonia. H. influenzae is sometimes considered to be an opportunistic pathogen as invasion usually follows a loss of the mucocilliary escalator function following either viral or toxigenic denudement. (Swords, W. E., M. R. Ketterer, J. Shao, C. A. Campbell, J. N. Weiser, and M. A. Apicella. 2001. Binding of the non-typeable Haemophilus influenzae lipooligosaccharide to the PAF receptor initiates host cell signalling. Cell. Microbiol. 3:525-536, incorporated by reference herein.) H. influenzae is associated with both acute and chronic infections for which it has developed specialized survival strategies.
The entire genome of the less virulent laboratory reference strain of H. influenzae, designated Rd, was sequenced and published in 1995 (Fleischmann et al., Science 269:496-512). However, since many of the genes that are responsible for the virulence of H. influenzae are missing from strain Rd, the sequence of the Rd genome is insufficient for developing tools to detect or prevent H. influenzae infections.
H. influenzae strains are classified according to their expression, or lack of expression, of a polysaccharide capsule. Encapsulated isolates are divided into six antigenic serotypes (a-f), whereas nonencapsulated isolates are classified as nontypeable H. influenzae (NTHi). H. influenzae type b (Hib) is associated with invasive disease and was previously responsible for the vast majority of H. influenzae-associated cases of meningitis. The introduction of an Hib vaccine in 1985 greatly decreased the incidence of type b infections (Scheifele, D. W., T. P. Jadavji, B. J. Law, R. Gold, N. E. Macdonald, M. H. Lebel, E. L. Mills, P. Dery, S. A. Halperin, R. F. Morris, V. Marchessault, and P. J. Duclos. 1996. Recent trends in pediatric Haemophilus influenzae type b infections in Canada. Can. Med. Assoc. J. 154:1041-1047; Schulte, E. E., G. S. Birkhead, S. F. Kondracki, and D. L. Morse. 1994. Patterns of Haemophilus influenzae type b invasive disease in New York State, 1987-1991: the role of vaccination requirements for day-care attendance. Pediatrics 94:1014-1016, both of which are incorporated by reference herein); however, non-type b strains, including the NTHi, continue to be important pathogens worldwide.
Most strains of H. influenzae are naturally competent, with the ability to take up DNA from their environments and integrate it into their chromosomes. Many naturally competent bacteria such as Haemophilus sp. and Neisseria sp. preferentially take up DNA from highly related organisms, which they recognize by the presence of genera-specific conserved uptake signal sequences (USSs) that are over-represented in their respective genomes (Elkins, C., C. E. Thomas, H. S. Seifert, and P. F. Sparling. 1991. Species-specific uptake of DNA by gonococci is mediated by a 10-base-pair sequence. J. Bacteriol. 173:3911-3913; Mathis, L. S., and J. J. Scocca. 1982. Haemophilus influenzae and Neisseria gonorrhoeae recognize different specificity determinants in the DNA uptake step of genetic transformation. J. Gen. Microbiol. 128:1159-1161; Smith, H. O., J.-F. Tomb, B. A. Dougherty, R. D. Fleischmann, and J. C. Venter. 1995. Frequency and distribution of DNA uptake signal sequences in the Haemophilus influenze Rd genome. Science 269:538-540, all of which are incorporated by reference herein). The H. influenzae Rd genome contains 1465 copies of the hUSS (Smith, H. O., J.-F. Tomb, B. A. Dougherty, R. D. Fleischmann, and J. C. Venter. 1995. Frequency and distribution of DNA uptake signal sequences in the Haemophilus influenze Rd genome. Science 269:538-540, incorporated by reference herein); thus, on average, there is about one hUSS per 1200 bases, or approximately one/gene. This frequency of the hUSS would theoretically provide for the exchange of any H. influenzae gene among strains. Thus, the natural transformation system of H. influenzae provides a mechanism for generating multiple types of genetic diversity among strains, including genetic heterogeneity (allelic differences) and genomic plasticity (genic differences).
There is evidence that inter-species transfers occur as well. Kroll et al. (Kroll, J. S., K. E. Willcs, J. L. Farrant, and P. R. Langford. 1998. Natural genetic exchange between Haemophilus and Neisseria: intergeneric transfer of chromosomal genes between major human pathogens. Proc. Natl. Acad. Sci. USA 95:12381-12385, incorporated by reference herein) have presented evidence of uptake sequence-mediated intergeneric transfer between the upper respiratory pathogens Haemophilus and Neisseria. These researchers identified three regions of Haemophilus-like DNA in the Neissera meningitidis genome and suggested that transformation with heterologous DNA may play an important role in establishing chromosomal mosaicism in these organisms.
Several studies have shown that chronic NTHi infections display significant genetic and phenotypic diversity (Loos, B. G., J. M. Bernstein, D. M. Dryja, T. F. Murphy, and D. P. Dickinson. 1989. Determination of the epidemiology and transmission of nontypeable Haemophilus influenzae in children with otitis media by comparison of total genomic DNA restriction fingerprints. Infect. Immun. 57:2751-2757; Porras, O., D. A. Caugant, B. Gray, T. Lagergard, B. R. Levin, and C. Svanborg-Edén. 1986. Difference in structure between type; van Alphen, L., D. A. Caugant, B. Duim, M. O'Rourke, and L. D. Bowler. 1997. Differences in genetic diversity of nonencapsulated Haemophilus influenzae from various diseases. Microbiology 143:1423-1431, all of which are incorporated by reference herein). van Alphen et al. used multilocus enzyme electrophoresis to examine the genetic diversity among 80 isolates of NTHi recovered from both healthy patients and those presenting with different diseases. These researchers concluded that chronic persistence in the host contributes to the genetic diversity of NTHi.
To examine the degree of genomic plasticity among pathogenic strains of H. influenzae, a highly redundant, pooled genomic library was generated using hydrodynamically sheared DNA from 10 clinical isolates cultured from pediatric patients undergoing treatment for chronic otitis media with effusion. This library is also being used as part of a larger investigation to study gene expression in order to delineate the genetic control of biofilm formation in pathogenic strains of H. influenzae. The present invention addresses the objective of evaluating the genomic plasticity between the nonpathogenic H. influenzae reference strain Rd and 10 clinical isolates, and DNA sequence evidence that reveals an extensive degree of plasticity among all strains.
The present invention involves isolated and cloned novel DNA fragments that are present in pathogenic strains of Haemophilus influenzae (cultured from pediatric patients undergoing treatment for chronic otitis media with effusion) and are absent in strain Rd. Four of these sequences are disclosed, whose corresponding amino acid sequences display varying degrees of homology to virulence-associated bacterial proteins.